H. Shinohara, N. Nagi-Miura, K. Ishibashi, Y. Adachi, A. Ishida

1854
Biol. Pharm. Bull. 29(9) 1854—1861 (2006)
Vol. 29, No. 9
Beta-Mannosyl Linkages Negatively Regulate Anaphylaxis and Vasculitis
in Mice, Induced by CAWS, Fungal PAMPs Composed of Mannoprotein–
Beta-Glucan Complex Secreted by Candida albicans
Hiroyasu SHINOHARA,a Noriko NAGI-MIURA,a Ken-ichi ISHIBASHI,a Yoshiyuki ADACHI,a
Akiko ISHIDA-OKAWARA,b Toshiaki OHARASEKI,c Kei TAKAHASHI,c Shiro NAOE,c Kazuo SUZUKI,b and
Naohito OHNO*,a
a
Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and
Life Science; 1432–1, Horinouchi, Hachioji, Tokyo 192–0392, Japan: b Department of Bioactive Molecules, National
Institute of Infectious Diseases; Tokyo 162–8640, Japan: and c Department of Pathology, Ohashi Hospital, Toho University
School of Medicine; Tokyo 153–8515, Japan.
Received April 19, 2006; accepted June 22, 2006; published online June 23, 2006
Candida albicans water soluble fraction (CAWS) is a water-soluble extracellular mannoprotein–beta-glucan
complex obtained from the culture supernatant of Candida albicans, which grows in a chemically defined
medium. CAWS induced toxic reactions, such as acute anaphylactoid reaction, by intravenous administration
and coronary arteritis by intraperitoneal administration. To clarify the structure responsible for these toxic reactions, C. albicans was cultured in pH- and temperature-controlled conditions and prepared with CAWS with or
without the beta-1,2-linked mannosyl segment (BM). The structure of CAWS was assessed by immunochemical
and spectroscopic methodologies, and we found that CAWS prepared under the natural culture conditions contained only small amounts of BM and CAWS prepared at neutral conditions at 27 °C contained a significantly
higher percentage of BM. Both the acute lethal toxicity and coronary arteritis induction was significantly more
severe in the absence of BM. Activation of a complement pathway, the lectin pathway, by CAWS was significantly
stronger in the absence of BM. These facts strongly suggest that BM linkages in CAWS negatively modulate
acute and chronic toxicity of CAWS, and may be strongly related to the lectin pathway of the complement activation.
Key words
Candida albicans; arteritis; mannoprotein; beta-1,2-mannan; DBA/2 mice
Candida spp. is a medically important fungus that frequently induces deep mycosis and fungemia, especially in
immunocompromised hosts.1) Pathogenic microbes have various toxic mechanisms that are unique to each species and
are essential for survival and evolution. Pathogenic mechanisms of eukaryotic microbes are complicated compared to
those of bacteria and viruses. Although there have been extensive basic and clinical studies, the major pathogenic factors for Candida spp. are still unknown and precise characterization of each of the constituents would be valuable.
Cardiovascular disease is one of the most life-threatening
diseases, and various new compounds and new methods of
pharmaceutical care, such as gene therapy and drug delivery
systems, have been developed to treat it. There are several
lines of evidence suggesting that inflammatory cells, particularly macrophages and neutrophils, regulate endothelial cell
function and dysfunction in atherosclerosis, via the release of
mediators that display proinflammatory activity. Analysis of
such diseases by animal models will give promising feedback
to human health.
Murata et al.2) reported that Kawasaki-disease-like coronary arteritis was induced in mice by administration of an alkaline extract of C. albicans (CADS) isolated from patients
with Kawasaki disease (KD). KD, a disease of unknown
cause that mainly affects children aged 4 and under, was first
reported by Kawasaki in 1967.3) The patient presents with
systemic coronary arteritis in nearly 10% of cases. Although
the occurrence of such coronary artery disorders has decreased with g -globulin therapy, the mechanism of occurrence along with the pharmacological mechanism of treat∗ To whom correspondence should be addressed.
ment is unknown.4) Murata’s murine model has been extensively examined from various points of view, e.g. antimyeloperoxidase antibody production, susceptibility loci,
and histopathological features.5,6)
Previously, we prepared a water-soluble polysaccharide
fraction of C. albicans released into a culture supernatant
(Candida albicans water-soluble fraction : CAWS) and performed various analyses.7—10) The most important point concerning this system is that we use a completely synthetic
medium, named C-limiting medium, to eliminate contaminants from the culture medium, such as endotoxins, peptidoglycans, and nucleic acids. Series of our studies found that
CAWS shows various toxic activities, such as cytokine synthesis by leukocytes, platelet aggregation, lethal toxicity, enhancement of side effect of indomethacin, induction of coronary arteritis in mice, and so on. Thus, CAWS may be a
pathogen-associated microbial product (PAMPs) of Candida
albicans.
In a previous study we found that intraperitoneal administration of CAWS to mice induces coronary arteritis similar to
that induced by CADS.8—10) It is of note that CAWS-induced
arteritis showed significant strain dependency: The incidence
of arteritis was 100% in C57BL/6, C3H/HeN and DBA/2
mice, but only 10% in CBA/J mice. The coronary arteritis
observed in DBA/2 mice was the most serious, with the majority of mice expiring during the observation period.
The coronary arteritis induced by CAWS was accompanied by hypertrophy of the tunica intima, the rupture of elastic fibers and a diffuse invasion by lymphocytes, histiocytes,
fibroblasts, smooth muscle cells and eosinophils of vascular
e-mail: ohnonao@ps.toyaku.ac.jp
© 2006 Pharmaceutical Society of Japan
September 2006
endothelial cells and the regions surrounding blood vessels.
Based on such characteristics, the coronary arteritis induced
by CAWS was presumed to be proliferative granulomatous
coronary arteritis, and is clearly different from fibrinoid arteritis. In DBA/2 mice, it was observed to cover nearly the
entire periphery of the vessels, and those mice were considered to demonstrate the most virulent form of coronary arteritis.
Structure of yeast cell wall, such as in Saccharomyces
cerevisiae, Candida albicans, and Schizosaccharomyces
pombe, have been extensively examined and it was found that
the overall structure is quite complex and regulated by expression of multiple genes.11,12) According to the results of
NMR and biochemical analyses, CAWS was found to be
composed of a mannoprotein and b -glucan portion, which
are the main components of the C. albicans cell wall.8) The
mannoprotein region of Candida albicans is composed of
both N- and O-glycosyl linkages with alfa- and beta-linked
mannosyl residues.13,14) Phosphodiester linkages are also included. Mannan synthesis is regulated by several specific
glycosyl transferases.15—17) It is also an immunochemical determinant of Candida spp.18,19) It is of note that the structure
of mannan is modulated by various culture conditions, such
as temperature and pH.20,21) The structure of mannan is also
known to be modulated by various stresses. The structure of
mannan is a key molecule for recognition of Candida by host
biodefense system and might be strictly regulated by the
affinity for specific receptors. Analysis of the disease from
the structural point of view is an indispensable study.
In a preliminary experiment, the pH of a culture medium
was measured and it was found that shifted from 5.2 to 2.3 at
the end. In the present study, we have prepared CAWS using
different culture conditions and examined CAWS-induced
anaphylactoid reaction and vasculitis. We demonstrated that
beta-1,2-mannosyl linkages are the negative regulators for
both anaphylactoid reaction and vasculitis.
MATERIALS AND METHODS
Mice Male ICR and DBA/2 mice were purchased from
Japan SLC. The mice were housed in a specific pathogenfree (SPF) environment and were used in the study at 5
weeks of age. All animal experiments were followed by the
guideline of laboratory animal experiments in Tokyo University of Pharmacy and Life Sciences (TUPLS), and each of
the experimental protocol was approved by the committee of
laboratory animal experiments in TUPLS.
Preparation of CAWS Candida albicans strain
IFO1385 was purchased from the Institute for Fermentation,
Osaka (IFO), stored at 25 °C on Sabauroud’s agar (Difco,
U.S.A.) and passaged once every three months. CAWS was
prepared from C. albicans strain IFO1385 in accordance
with conventional methods.7,9,10) The procedure used was as
follows: 5 l of medium (C-limiting medium) was added to a
glass incubator and cultured for 2 d at 27 °C with air supplied
at a rate of 5 l/min and rotation at 400 rpm. Following the
culture, an equal volume of ethanol was added and after the
mixture was allowed to stand overnight, the precipitate was
collected. The precipitate was dissolved in 250 ml of distilled
water, ethanol was added and the mixture was allowed to
stand overnight. The precipitate was collected and dried with
1855
acetone to obtain CAWS.
Administration Schedule for Induction of Coronary
Arteritis (10) CAWS (4 mg/mouse) was administered intraperitoneally for 5 consecutive days to each mouse in week
1. At week 5, the hearts of the animals were fixed with 10%
neutral formalin and prepared in paraffin blocks. Tissue sections were stained with hematoxylin–eosin (HE) stain.
ELISAs of Candida Typing Sera to CAWS The reactivity of CAWS to serum factors from Candida that consists
of rabbit polyclonal antibodies against Candida cell wall
mannan was detected by ELISA. A solution of CAWS in
50 mM carbonate buffer (pH 9.6) was coated onto Nunc immunoplates, which were then incubated at 4 °C overnight.
The plates were washed extensively with 0.05% Tween 20
containing PBS (PBST); unbound sites were blocked by the
addition of 1% BSA containing PBST (BPBST) to wells for
40 min at 37 °C and then the wells were washed 6 times with
PBST. Candida serum factors serially diluted with BPBST
were added, and incubated for 60 min at 37 °C. After 6
washes with PBST, the wells were treated with peroxidaseconjugated goat anti-rabbit IgG and the 3,3,5,5-tetramethylbenzidine microwell peroxidase substrate system (TMB;
KPL inc). After 45 min, the reaction was stopped with 1 N
H3PO4, and then the optical density of each well was read at
450 nm on an automatic microplate reader.
Measurement of Complement Activation The complement kit for assessment of classical, alternative and MBL
pathway activity was developed by the EU consortium and is
now commercially available (Wielisa COMPL300 Total
Complement Functional Screen kit from Wieslab AB, Lund,
Sweden). The kit was operated by the instructions provided
in the manual with slight modifications. In brief, strips of
wells for classical pathway (CP) evaluation were precoated
with IgM, strips for alternative pathway (AP) determination
were coated with LPS, and mannose binding lectin (MBL)
pathway (MBL-P) strips were coated with mannan. Sera
were diluted 1/101 for the CP and MBL-P assay and 1/18 for
the AP assay in specific buffers, serial dilutions of CAWS solution was added to the diluted sera and incubated. The resulting sera were added to the strips of the kit and were incubated for 1 h at 37 °C. After washing the strips, alkaline
phosphatase-conjugated antihuman C5b-9 was added and incubated at room temperature for 30 min. Additional washing
was performed, substrate was added, and the wells were incubated for 30 min. Finally, absorbance values were read at
405 nm. In each assay, standard positive and negative control
sera provided in the kit as lyophilised material were reconstituted with distilled water. Complement activity was calculated using the following formula: activity100%(mean
A405 (sample)mean A405 (negative control)/(mean A405
(standard serum)mean A405 (negative control). Samples as
well as standard serum and negative control serum were
tested in duplicate at a fixed dilution.
Assay for Anaphylactoid Reaction Indicated dose of
CAWS solution was i.v. administered to ICR mice. The incidence and the severity of the rapid anaphylactoid shock were
assessed within 30 min. For measuring tolerance, low dose
CAWS was i.v. administered to ICR mice 1 h prior to induce
anaphylactoid reaction by CAWS (400 m g/mouse).
NMR Spectroscopy H1-NMR experiments were performed with a Bruker DPX 400 equipped with XWIN-NMR
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Vol. 29, No. 9
software. The spectra were recorded using a solution of each
soluble fraction (10 mg/ml) in D2O at 45 °C with acetone as
an internal standard.
RESULTS
Preparation of CAWS by Various Culture Conditions
Figure 1 shows the data of pH monitoring during Candida
culture in C-limiting medium. The data clearly demonstrated
that at pH uncontrolled natural time course, pH was 5.2 at
the beginning and was lowered to around 2 at the end, thus
the naturally expressed CAWS was produced in this culture
condition. In order to prepare CAWS with modulated mannan structure, C. albicans IFO1385 was cultured at either
27 °C or 37 °C in C-limiting medium. In addition, pH of the
culture medium was controlled at either pH 2.3, 5.2, or 7.0
by automatically added sodium hydroxide solution. The extracellular mannoprotein fraction was prepared by a previously established procedure that was the same as naturally produced CAWS. The names of each fraction were
designated as CAWS 27-2.3, CAWS 27-5.2, CAWS 27-7.0,
CAWS 37-2.3, CAWS 37-5.2, and CAWS 37-7.0. The name
of CAWS prepared at natural pH course was designated as
27-(–) and 37-(–), respectively. In addition, CAWS prepared
at 27 °C was designated as CAWS 27s which include 27-2.3,
27-5.2, 27-7.0, and 27-(–), and prepared at 37 °C as CAWS
37s which include 37-2.3, 37-5.2, 37-7.0, and 37-(–). Yield
and some parameters of each CAWS are shown in Table 1.
The structure of the mannan moiety was characterized by the
reactivity to the typing sera and NMR spectral analysis.
Figure 2 shows relative reactivity to the typing sera no. 5,
6, and 11. Experiments were done using three doses of
CAWS solution adsorbed onto the ELISA plates. The pattern
of reactivity of each CAWS preparation to the sera were similar between no. 5 and no. 6. In contrast, reactivity to no. 11
was significantly different. In the cases of no. 5 and no. 6,
comparing the dose to show absorbance 1.0, reactivity of
CAWS 27-5.2 and CAWS 27-7.0 was significantly increased
compared to CAWS 27-(–), and other preparations. In contrast, relative activity of CAWS 27-5.2 and CAWS 27-7.0
Fig. 1. Monitoring pH of C. albicans IFO1385 Culture in C-Limiting
Medium at 27 °C
Fig. 2. Reactivity of CAWS Prepared under Various Conditions to Candida Typing Sera no. 5, 6, and 11
One hundred milliliters of C. albicans IFO1385 precultured in liquid to logarithmic
phase was added to 4 l of C-limiting medium in a jar fermenter with 4 l air/min and
400 rpm stirring. The pH of the culture medium was measured automatically with a pH
meter.
Each 25, 2.5, or 0.25 m g/ml solution of CAWS was adsorbed on ELISA plate and diluted typing sera was added. After appropriate incubation time, plate-bound antibody
was measured by peroxidase conjugated anti-rabbit IgG with TMB reagent. (A) Anti
no. 5; (B) anti no. 6; (C) anti no. 11.
Table 1.
Properties of CAWS Prepared by Various Culture Conditions
CAWS
Yield (mg/l)
Cell (g/l)
C (%)
H (%)
N (%)
Factor G
Factor C
27-(–)
27-2.3
27-5.2
27-7.0
37-(–)
37-2.3
37-5.2
37-7.0
14730
13219
24726
38177
8433
16017
25587
26312
4.30.7
4.00.2
3.50.4
3.40.1
2.00.7
3.30.1
3.00.2
3.20.5
30.12.7
27.54.7
28.63.5
18.15.3
26.51.8
18.59.1
20.32.7
24.31.1
5.50.4
5.00.7
5.20.4
4.00.8
4.80.4
3.71.5
4.00.7
4.90.1
1.60.3
1.60.1
2.80.3
2.10.6
1.10.1
1.10.2
0.90.2
1.50.1
—
0.730.57
ND
ND
1.240.21
0.560.16
ND
0.070.01
<2
0.250.1
0.330.45
0.710.73
0.030.02
ND
0.10.03
0.080.01
27-(–); cultured at 27 °C without pH control, 27-2.3; cultured at 27 °C and pH was maintained at 2.3, 27-5.2; cultured at 27 °C and pH was maintained at 5.2, 27-7.0; cultured
at 27 °C and pH was maintained at 7.0, cell; yield of dried cells. C, H, N; elemental analysys (carbon, hydrogen, nitrogen), factor G; content of 1,3-beta-glucan (ng/ml), factor C;
content of endotoxin (ng/ml), ND; not detected.
September 2006
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Table 2. Anaphylactoid Reaction Induced by CAWS Prepared under Various Conditions
CAWS
27-(–)
27-2.3
27-5.2
27-7.0
37-(–)
37-2.3
37-5.2
37-7.0
3200
2/3
0/3
800
0/3
0/3
200
50
3/3
3/3
0/3
0/3
2/3
3/3
2/3
2/3
0/3
0/3
1/3
1/3
0/3
0/3
12.5
0/3
0/3
Indicated dose (m g/mouse) of CAWS was i.v. administered to mice (n3). Lethality
of each mouse was monitored 30 min later. ∗/∗, number of mice, dead/total.
Fig. 3.
Proton-NMR Spectra of CAWS in D2O
(A) CAWS 27s prepared from the 27 °C culture. (B) CAWS 37s prepared from the
37 °C culture. Arrow indicates signals corresponding to beta-1,2-mannosyl linkages.
was significantly decreased in the case of typing sera no. 11.
It is proposed that the specific epitope of no. 5 and no. 6 are
for the beta-1,2-linked mannooligosaccharides, and no. 11
for the alpha-1,2 and 1,6-mannooligosaccharides.20,21) These
data strongly suggested that CAWS 27-5.2 and CAWS 27-7.0
contained significantly higher concentration of beta-1,2-linkages and reduced concentration of alfa-linkages.
Figure 3 shows NMR spectra of CAWS preparations. Each
of the spectra contained many signals in the anomeric region
(4.8—5.5), and thus, we could not do complete assessment at
this time. However, comparing the published spectra of the
cell wall mannan moiety of Candida spp. by Professor
Suzuki et al.,20,21) major signals could be assigned. Chemical
shift around 4.85, shown as arrow in the spectra, would be
beta-linked manno-oligosaccharides. The signal around 4.85
was only detected in the spectra of CAWS 27-5.2 and CAWS
27-7.0. Signals around 5.13 also appeared in those spectra
would be the residues linked to beta-mannosyl linkages. In
contrast, signals around 5.05 which include alpha-mannose
residues was reduced in relation to the substitution of betamannosyl linkages. It is of note that even in the same pH
conditions, CAWS 37-5.2 and CAWS 37-7.0 did not show
such signals, and this observation was strongly supported by
immunochemical reactivity. NMR spectra of CAWS 37-(–),
CAWS 37-2.3, CAWS 37-5.2, and CAWS 37-7.0, shown in
Fig. 3b show simple anomeric signals compared to those of
CAWS 27s. Shibata et al. examined the cell wall mannan
structure of Candida parapsilosis and Candida albicans by
use of controlled hydrolysis and NMR spectroscopy.22) Comparing Proton NMR spectra of C. parapsilosis and C. albicans, that of C. parapsilosis is simple and shows only 5
major signals in the anomeric region of 1D-NMR spectrum.
Those signals correspond to alpha mannosyl linkages. At
least in 1D-NMR spectrum, CAWS 37s resembles that of C.
parapsilosis, also supporting that beta-1,2-linkages are missing in CAWS 37s. Regulation of beta-1,2-mannosyl linkages
by culture condition is also reported in Candida guilliermondii.23) From the data of immunochemistry and NMR
spectra, CAWS 27-5.2 and CAWS 27-7.0 contained significantly higher concentrations of beta-1,2-linked mannooligosaccharides.
Acute Toxicity of CAWS Prepared by Various Culture
Conditions We have previously shown that CAWS induced
acute anaphylactoid reaction, similar to E. coli O9 LPS and
yeast mannan.24,25) We compared acute anaphylactoid reaction of various CAWS preparations. As shown in Table 2,
various concentrations of CAWS were intravenously administered to ICR mice and survival within 30 min was compared. In this experiment, the majority of CAWS preparations induced almost complete lethal toxicity at doses of
200 m g/mouse. However, CAWS 27-5.2 required 3200 m g for
lethal toxicity and CAWS 27-7.0 did not show any lethal toxicity in this experimental condition. These facts strongly suggested beta-1,2-linked manno-oligosaccharide would be a
negative regulator for acute anaphylactoid reaction. To confirm the possibility, tolerance induction by CAWS preparations was also tested. As shown in Table 3, administration of
16 m g of CAWS 27-(–) and CAWS 27-2.3 significantly reduced lethality of subsequent high dose CAWS administration. However, in the case of CAWS 27-5.2, a relatively
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Table 3. Tolerance Induction in CAWS Induced Anaphylactoid Reaction
by Pretreatment with CAWS Prepared under Various Conditions
CAWS
27-(–)
27-2.3
27-5.2
64
16
4
1
3/3
2/3
2/3
2/3
0/3
1/3
0/3
2/3
Indicated dose of CAWS (m g/ml) was i.v. administered to mice (n3). CAWS-(–)
was again i.v. administered to each mouse, and lethality was monitored 30 min later.
∗/∗, number of mice, dead/total.
Fig. 5.
Histological Analysis of DBA/2 Mice Administered with CAWS
CAWS prepared under various conditions (4 mg/mouse) was administered i.p. to
DBA/2 mice for five consecutive days. Five weeks later, the aorta with coronary of
these mice were stained with hematoxylin–eosin.
Fig. 4.
Complement Activation by CAWS
(A) Screening of classical (CP), lectin (MP), and alternative (AP) pathway of complement activation by CAWS. (B) Dose response of CAWS-induced activation of the
lectin pathway of complement. CAWS 27-(–) and CAWS 27-7.0 were used in this experiment. PC; positive control serum, NC; negative control serum.
higher dose, 64 m g, was required for tolerance induction.
This result also strongly suggested that beta-1,2-mannooligosaccharides negatively modulate acute lethal toxicity of
CAWS.
Complement activation and the resulting anaphylactic peptides, C3a and C5a, production are the most commonly postulated mechanism for acute toxicity. In the preliminary experiments, incubation of CAWS with human sera produced
high concentration of C5a anaphylatoxic peptide. Because of
the difficulty in measuring mouse complement activation, we
used a commercially available kit for human complement activation in the present study. Activity of classical, lectin, and
alternative pathways were measured using IgM, mannan, and
LPS-coated ELISA plate and specific reaction buffer systems. As shown in Fig. 4a, CAWS of 20 to 2000 m g/ml were
added, and it was found that CAWS activated complement
was almost specifically mediated by the lectin pathway. In
this dose range, CAWS 27-(–) and CAWS 27-7.0 did not
show any differences; however, comparing the activity be-
tween doses of 20 ng and 200 ng shown in Fig 4b, CAWS 27(–) showed almost ten times higher activity than CAWS 277.0. These results strongly suggest that beta-1,2-linked mannooligosaccharides inhibited complement activation by alfalinked mannose residues.
Arteritis Induction by CAWS Prepared by Various Culture Conditions Intraperitoneal injection of CAWS induced coronary arteritis in mice.8—10) Severity is significantly
dependent on strains of mice and DBA/2 shows the most severe reaction, resulting in death. Coronary arteritis induction
by CAWS prepared as above was compared. CAWS was administered i.p. to each mouse at 4 mg/mouse for 5 consecutive days and sections of coronary artery were prepared at 4
weeks after the final CAWS administration. Figures 5 to 8
show hematoxylin eosin staining of aorta, aortic valve, and
the coronary artery in CAWS-administered mice. CAWS prepared by the standard protocol induced strong inflammation
in this area. CAWS 27-2.3, CAWS 37-(–), CAWS 37-2.3,
CAWS 37-5.2, and CAWS 37-7.0 show similar inflammation.
However, CAWS 27-5.2 and CAWS 27-7.0, both of which
contained higher content of beta-1,2-manno-oligosaccharides, show significantly weaker inflammation. The histology
of the aorta, aortic valve, and coronary artery using higher
magnification (Figs. 7, 8) shows similar phenotypes.
Coronary arteritis was also examined by survival. As
shown in Fig. 9, almost all of the mice that had CAWS administered died except for CAWS 27-5.2, in which only one
mouse out of five died. Figure 10 shows the dose response of
survival and corresponding histology of DBA/2 mice administered CAWS 27-(–). Arteritis was induced even with
250 m g/mouse administration and 2/5 mice died in this condition. Considering the data of survival and histology of
CAWS 27-(–) and CAWS 27-5.2, the relative activity of
September 2006
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Fig. 6. Comparison of Histology of Aorta in DBA/2 Mice Administered
Native and pH-Controlled CAWS
CAWS 27-(–), CAWS 27-5.2, CAWS 37-(–), CAWS 37-5.2 (4 mg/mouse) were administered i.p. to DBA/2 mice for five consecutive days. Five weeks later, the aorta with
coronary of these mice were stained with hematoxylin–eosin.
Fig. 8. Comparison of Histology of Coronary Artery in DBA/2 Mice Administered Native and pH-Controlled CAWS
CAWS 27-(–), CAWS 27-5.2, CAWS 37-(–), CAWS 37-5.2 (4 mg/mouse) were administered i.p. to DBA/2 mice for five consecutive days. Five weeks later, the artery
with coronary of these mice were stained with hematoxylin–eosin.
Fig. 9.
Survival Time of CAWS-Administered Mice
CAWS 27s and CAWS 37s (4 mg/mouse) was administered i.p. to DBA/2 mice for
five consecutive days in the 1st week. Survival was observed for 12 weeks.
Fig. 7. Comparison of Histology of Cusp Region of Coronary Artery in
DBA/2 Mice Administered Native and pH-Controlled CAWS
CAWS 27-(–), CAWS 27-5.2, CAWS 37-(–), CAWS 37-5.2 (4 mg/mouse) were administered i.p. to DBA/2 mice for five consecutive days. Five weeks later, the aorta with
coronary of these mice were stained with hematoxylin–eosin.
CAWS 27-(–) was at least 20 times higher than that of
CAWS 27-5.2. From these findings, CAWS with beta-1,2linked mannooligosaccharides showed significantly weaker
inflammatory reaction in this experimental model.
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Fig. 10.
Vol. 29, No. 9
Dose Response of Survival Time of CAWS-Administered Mice
CAWS 27-(–) (0.25, 1, or 4 mg/mouse) was administered i.p. to DBA/2 mice for five
consecutive days in the 1st week. Survival was observed for 9 weeks.
Hematoxylin–eosin stain of the aorta with coronary were also shown.
DISCUSSION
Candida albicans is a clinically important fungus and is
known to cause disseminated candidiasis and candidemia in
immunocompromised hosts. Patients with deep mycoses,
such as that induced by C. albicans and A. fumigatus, have
been demonstrated to release b -glucans into the blood. However, these are present in extremely small amounts, and the
overall structure of the factor G activating substance present
in the blood is unknown.26) The factor G activating substance
has the potential to exhibit various biological activities, but
the details of this are also unknown. In order to clarify these
matters, we first cultured C. albicans in a completely synthetic medium in order to obtain water-soluble limulus factor
G activating substance that is released from the cells, and obtained a water-soluble polysaccharide fraction released into
the culture supernatant (CAWS), which is thought to be similar to the b -1,3-D-glucan actually present in patient blood.8)
Murata et al. conducted an analysis on children with KD and
found that C. albicans extract (CADS) isolated from the
stool specimens of the patients induced coronary angiitis in
mice that resembled KD.5,6) During the course of joint research, we found that administration of CAWS according to
this protocol induced a similar coronary angiitis in mice.8—10)
In the present study, the relationship between culture conditions of Candida and induction of some of the pathologic
parameters, such as acute shock, arteritis, and complement
activation, was examined, and the condition producing beta1,2-linkage significantly reduced all three parameters measured in the present study. It is of note that in the standard culture medium, C-limiting medium, the pH was acidified during culture. Thus, in the present study we have prepared
CAWS in significantly regulated conditions and we analyzed
both activity and structure. We have confirmed that CAWS
prepared under the condition of neutral at 27 °C culture significantly reduced activity and is strongly related to the presence of beta-1,2-mannosyl linkages. These facts clearly
demonstrated that beta-1,2-linkages are negative regulators
for pathologic parameters, such as acute shock, arteritis, and
complement activation. At this time, the role of beta-1,2linkages on the anaphylactoid reaction and arteritis induction
could not be fully clarified. As shown in Table 3, pretreatment of mice with CAWS 27-5.2 induced tolerance in the
CAWS-induced anaphylactoid reaction, but this was less than
that observed with CAWS 27-(–) and CAWS 27-2.3. In addition, small numbers of beta-1,2-linkages are present in
CAWS 27-(–), which is comparable arteritis to CAWS 272.3. These facts strongly suggest that beta-1,2-linkages cover
the active site, the alpha mannan moiety, of CAWS. Beta-1,2linkage itself might not show receptor-mediated, specific and
negative regulatory reactions.
Acute anaphylactoid reaction is known to be induced by
lipopolysaccharide from E. coli O9 (O9 LPS), which possesses the mannose homopolysaccharide as the O-antigen region.25) Very recently, we compared immunotoxicological
and immunochemical similarity between CAWS and O9
LPS.24) CAWS strongly reacted with Candida serum factors,
and the reactivity was found to be partially competed with
O9 LPS. CAWS-induced anaphylactoid reaction was inhibited by pretreatment of mice with i.p. injection of CAWS.
The lethality was found to be inhibited by i.v. injection of O9
LPS. Vice versa, O9 LPS-induced acute anaphylactoid reaction in muramyl-dipeptide primed mice was also inhibited by
pretreatment of mice with CAWS. These results suggested
that CAWS and O9 LPS from gram-negative bacteria share,
at least in part, immunochemical and immunotoxicological
activities.
Mannose binding lectin (MBL) is a key molecule for innate immune response.27) Neth et al. analyzed the concentration of MBL in various inbred strains and found that all of
the strains tested contained MBL.28) The MBL-A and MBLC levels in 10 laboratory mice strains were 4 to 12 m g/ml,
and 16 to 118 m g/ml, respectively. Higher concentration of
MBL was detected in patients of rheumatic heart disease.29)
Point mutation in MBL is also related to severe atherosclerosis.30) In the present study, we have shown contribution of the
complement lectin pathway for the initiation of CAWS-induced anaphylactoid reaction and arteritis. We did not measure concentration of each complement components, and concentration of each component in inbred strains might be related to the phenotype of anaphylactoid reaction and arteritis.
The anaphylatoxin receptor is a family of G-protein coupled receptors (G-PCR).31) It is well known that signaling of
G-PCR induced cross talk and cross desensitization.32) Cross
desensitization of C5a receptor was reported with receptors
of fMLP, IL-8, LTB4 and so on. Exaggerated inflammatory
phenotype of bleomycin-induced lung fibrosis was shown in
C5 knockout mice. DBA/2 is a strain deficient in C5. Severity of arteritis might be related to cross desensitization and
may follow anti-inflammatory action of C5a receptor mediated signaling.
In the series of studies, we have analyzed CAWS-induced
arteritis from various points of view. The most striking result
was the significant strain dependency of arteritis as well as
acute anaphylactoid reaction. For example, C3H/HeN is the
most sensitive strain for anaphylactoid reaction but arteritis
is moderate. DBA/2 is completely resistant to anaphylactoid
reaction but shows the most severe arteritis. CBA/J is the
September 2006
most resistant strain for arteritis but induced anaphylactoid
reaction (unpublished results). B6 is sensitive to both anaphylactoid reaction and arteritis. These facts strongly suggest
that the genes responsible for anaphylactoid reaction and arteritis would be at least in part different, and thus regulated
by multiple genetic factors. However, from the point of early
signaling events, activation of the lectin pathway of the complement by alpha-mannan moiety of CAWS might be key for
both of the activities. Several reports indicated that Candida
albicans could bind to MBL.27,28)
We have shown that CAWS-induced arteritis and anaphylactoid reaction is dependent on strains of mice. In the present study, we demonstrated the essential structure to induce
arteritis and anaphylactoid reaction. Strain specificity of both
reactions was independent. We also analyzed the kinetics of
arteritis induction and determined that it is separated into at
least three phases, i.e., early, middle, and late.10) In the early
phase, a variety of biochemical events is induced. In the middle phase, remodeling of arteries is induced. In the late
phase, arteritis and coronary arteritis stressed heart function,
resulting in cardiomegaly. Undoubtedly, multiple genes are
involved in arteritis and cardiomegaly. This model proved
very useful for analyzing whole steps of cardiac diseases.
Acknowledgements This work was partly supported by a
Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology of Japan,
and The Promotion and Mutual Aid Corporation for Private
Schools, Japan. Also supported by Ministry of Health,
Labour and Welfare in Japan a Grant on “Research on Regulatory Science of Pharmaceuticals and Medical Devices.”
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